Accepted Manuscript

Creatine as a booster for human brain function. How might it work?

Prof Caroline D. Rae, Stefan Bröer

PII: S0197-0186(15)30038-3
DOI: 10.1016/j.neuint.2015.08.010
Reference: NCI 3758

To appear in: Neurochemistry International

Received Date: 14 July 2015

Revised Date: 4 August 2015
Accepted Date: 15 August 2015

Please cite this article as: Rae, P.C.D., Bröer, S., Creatine as a booster for human brain function. How
might it work?, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.08.010.

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 ACCEPTED MANUSCRIPT
Creatine as a booster for human brain function. How might it work?

Caroline D. Rae1,2 & Stefan Bröer3

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1. Neuroscience Research Australia, Barker St Randwick, NSW 2031, Australia

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2. School of Medical Sciences, UNSW, High Street, Randwick, NSW 2052, Australia

3. Research School of Biology, The Australian National University, Canberra, ACT

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0200, Australia

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Corresponding author: Prof Caroline Rae, Neuroscience Research Australia, Barker St
Randwick, NSW 2031, Australia Email: c.rae@unsw.edu.au
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Abstract

Creatine, a naturally occurring nitrogenous organic acid found in animal tissues, has been

found to play key roles in the brain including buffering energy supply, improving

mitochondrial efficiency, directly acting as an anti-oxidant and acting as a neuroprotectant.

Much of the evidence for these roles has been established in vitro or in pre-clinical studies.

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Here, we examine the roles of creatine and explore the current status of translation of this

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research into use in humans and the clinic. Some further possibilities for use of creatine in

humans are also discussed.

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Introduction

Creatine (2-[Carbamimidoyl(methyl)amino]acetic acid, also known as methylguanidoacetic

acid) is a naturally occurring nitrogenous organic acid found in animal tissues. Discovered in

1832 by Michel Chevreul and chemically identified in 1847 [1], the compound, isolated by

basification of muscle, was named “creatine”, derived from kreas (κρέας) the Greek word for

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“meat”. The identifier of creatine and one of the fathers of Biochemistry, Justus von Liebig,

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largely supported his research endeavours by making and selling Liebig’s meat extract

(Fleischbrühe in German) which contained about 8% creatine [2]; plainly even the very first

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scientists working with creatine recognised its potential as a supplement.

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Where does creatine come from? AN
Humans obtain creatine from two sources; by consumption (of meat or artificial sources such

as laboratory-synthesised creatine) and by synthesis. Creatine is taken up from the gut by the
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sodium dependent creatine transporter (see below for more discussion of transport
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mechanisms) but it is not clear how it crosses the basolateral membrane [3].
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Brain synthesises creatine, although the majority of total body synthesis takes place in

kidney, pancreas and liver. Creatine is synthesised in a two-step reaction (Fig. 1); the amidino
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group is transferred from arginine to glycine in a reaction catalyzed by L-arginine-glycine

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amidino transferase (AGAT: E.C. 2.1.4.1) forming guanidinoacetate. This molecule is

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subsequently methylated by transferring the methyl group from the donor S-adenosyl-L-

methionine, yielding creatine and S-adenosylhomocysteine in a reaction catalysed by

guanidinoacetate-methyltransferase (GAMT; E.C. 2.1.1.2). Deficiencies in either of these

enzyme activities cause developmental delay with mental retardation and language deficits

(Fig. 1). GAMT deficiency [4] results in a more severe phenotype than AGAT deficiency [5,

6], most probably due to the extra effects of elevated guanidinoacetate (GAA). These effects

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include intractable (to anti-epileptic medication) seizures and extrapyramidal motor signs.

GAA is an antagonist at GABAρ receptors [7] as are other guanidino compounds, which are

also elevated in GAMT-deficiency [8]. These particular symptoms can be reduced by

restricting arginine intake and thus reducing GAA levels. Both deficiencies can be

remediated by supplementation with significant amounts (300 – 400 mg/kg/day [9]) of

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creatine.

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A third creatine deficiency disorder exists [10] caused by mutations in the sodium-dependent

creatine transporter, SLC6A8. While symptoms are in common with those caused by lack of

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creatine synthesising enzymes, the deficiency is mostly intractable to remediation with oral

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creatine or with precursors such as guanidinoacetate or combined arginine/glycine therapy
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[11]. Guanidinoacetate, the intermediate in creatine synthesis is also a substrate of SLC6A8.

Investigation of these disorders and why or why not they respond to creatine supplementation
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therapy has proven illuminating for our understanding of brain creatine synthesis and
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compartmentation in the brain. The creatine transporter SLC6A8 is an active, sodium-

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dependent creatine uptake mechanism; creatine concentrations in the brain are several fold

higher than in plasma (40 µmol in plasma vs 6-12 mM in brain) so there is clearly active
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accumulation of creatine. However, supplementation with oral creatine has only limited

effect on brain creatine; supplementation with 5 g/day for 6 weeks increased brain creatine by
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an average of only 11% in healthy young men [12], while ~20g/day for 7 days followed by 2
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g/day for 7 days resulted in increases of ~8% [13]. Studies using a shorter supplementation

time frame or doses lower than 5 g have tended to report no significant change in brain

creatine levels [14]. It has been suggested that the creatine transporter is near saturated

meaning its ability to increase uptake under physiological conditions is limited. Transfer of

creatine across the blood-brain and blood-CSF barrier appears to be limited [15] and must be

supplemented through creatine synthesis (Fig. 2).

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Cells in the brain have been shown to cooperate in creatine synthesis (Fig. 2). While some

cells in the brain have both synthesising enzymes and can make their own supply of creatine,

some cells have only AGAT and export guanidinoacetate which is taken up via SLC6A8 by

cells with GAMT and used to make creatine [10, 11]. Surprisingly GAMT is absent in many

neurons [16], while expressed at higher levels in oligodendrocytes and astrocytes. This

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suggests that glial cells may serve as local producers of creatine, which is then accumulated

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in neurons via SLC6A8. The bulk of brain SLC6A8 appears to be expressed in neurons [17],

small amounts in endothelial cells of the blood-brain barrier, but it appears to be absent from

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astrocytes [18].

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This means that the ability to take up either guanidinoacetate or creatine is crucial to
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maintain brain creatine supplies even when brain creatine synthesis machinery is intact and

explains why dysfunction of the creatine transporter SLC6A8 is so deleterious.

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The route by which creatine effluxes from cells - as proposed for astrocytes - is still unclear
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(marked unknown in Fig. 2). It is plain that creatine effluxes from cells at a steady rate and
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that normally this efflux sustained by sodium-dependent uptake or by endogenous synthesis.

Guanidinoacetate has been reported to leave the brain via the taurine transporter [15] . Other
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candidate efflux transporters might be the organic cation transporters (OCTs) which are

known to transport creatine (hOCT2), a range of guanidine compounds and creatinine [19].
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These transporters may also be responsible for creatine efflux from the basolateral membrane
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in the gut [20]. A recently discovered creatine transporter, SLC16A12, or MCT12 is not

expressed in the brain apart from eye [21] and is unlikely to be a candidate.

Creatine is also known to efflux from brain following osmotic challenge [22] but the route by

which it does this remains unclear. Astrocytes have been shown to have an efflux mechanism

for creatine which is pharmacologically distinct from that for taurine [23].

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It appears that under physiological conditions net import of creatine occurs through the blood

brain barrier, while net efflux occurs through choroid plexus [24]. Expression of SLC6A8

has been reported on the blood and brain side of blood brain barrier endothelial cells [25],

however, for a net blood to brain flux a different exit route must be proposed. Local synthesis

in astrocytes adds further creatine. The steady state amount of creatine is then accumulated

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into neurons through SLC6A8. Thus, there is an equilibrium between accumulation of

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creatine through SLC6A8 in the blood brain barrier and neurons and efflux of

creatine/guanidinoacetate/creatinine from the brain through blood-CSF barriers, for instance

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via organic cation transporters. Therefore, lack of SLC6A8 would drain the brain of creatine.

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Roles of creatine in the brain
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The primary and best recognised role of creatine is as an acceptor of high energy phosphate

in the reaction catalyzed by creatine kinase (Eq 1).

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Creatine + ATP ↔ PCr + ADP + H+ , (Eq 1).

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Creatine kinase mediates fast exchange between the energy currency ATP and the
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phosphorylated form of creatine, phosphocreatine. The reaction also requires a proton and so
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may change the local pH under conditions of high ATP demand [26, 27].

Phosphocreatine, through the creatine kinase-catalysed reaction, acts in concert with multiple
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ATP-producing and requiring reactions as a buffer for high energy phosphate bonds. The
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high energy phosphate bond in phosphocreatine has a higher free energy of hydrolysis than

that in ATP (∆G°′ kJ/mol = -45.0 cf. -31.8, respectively; [28]). Phosphocreatine is a smaller,

less negatively charged molecule than ATP, its diffusion to sites of demand is fast and

creatine kinase can quickly interconvert the two. Given ample PCr, the creatine kinase

reaction regenerates ATP at a rate 40 times faster than oxidative phosphorylation and 10

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times faster than glycolysis [29]. Phosphocreatine therefore acts as a quickly accessible

“Swiss Bank account” for energy currency, allowing cells to “hide” ATP in an accessible

form.

The brain is an organ with high energy demands, using around 20% of resting metabolism in

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adults despite accounting for only 2% of actual mass [30]. It is an energetically expensive

organ to run as a consequence and it has evolved to conserve energy with complex

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autoregulatory systems in place to rapidly upregulate metabolism once an area of the brain is

activated (for detailed discussion of mechanisms and their limiting bounds see [31]). These

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include rapid increases in blood flow and blood volume to deliver the extra oxygen and

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glucose that is required to fuel brain energy demands. Inspection of the time courses of these
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regulatory mechanisms shows blood flow peaking several seconds following a stimulus with

brain eventually reaching a new steady-state metabolism if the stimulus is continued [32, 33].
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This means that energy is delivered to the brain as and when it is required, incurring

significant energy savings, but the flipside to this is that the brain is not necessarily operating
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at maximal capacity when suddenly required to do a task. In the few seconds immediately
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following stimulation, brain physiological and biochemical responses are characterised by an

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“initial dip” (Fig. 3). Local brain oxygen supplies dip, as evidenced by reduction in the water

signal on fMRI [34] caused by a relative increase in paramagnetic deoxyhaemoglobin [35].

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Local glucose levels also dip although the length of the dip and recovery are on a much
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longer time scale than the localised dip in oxygen levels [36]. Studies using 31P magnetic

resonance spectroscopy (MRS), and also long echo time 1H MRS which is susceptible to

relative changes in the ratio of PCr to creatine (for discussion of measurement of creatine

using MRS see [37, 38]), have shown initial dips in the level of phosphocreatine in the

occipital cortex following photic stimulation [26, 27, 39, 40] along with increased local pH,

suggesting hydrolysis of PCr through the creatine kinase catalysed reaction (Eqn 1). It is

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likely that the pH changes reflect the activity of cytosolic creatine kinase; MRS is spatially a

relatively insensitive method and even less so with the 31P nucleus. The relative volume of

the cytosolic compartment compared to the mitochondrial one (around 250:1) means that it is

unlikely that changes in mitochondrial pH in the absence of changes in cytosolic pH would

be detectable [41].

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Although it is generally accepted that PCr and the creatine kinase reaction buffer ATP levels

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to the point where they are barely altered by brain activity [27] it is possible that they do

initially alter in healthy people if the brain is stimulated hard from a rested state [42]. It is

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also apparent that there are disorders and conditions where ATP levels do change with

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workload [43-45].
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The different isoforms of creatine kinase are key (Fig. 4). Brain contains the cytosolic brain-

type creatine kinase (BB-CK) [46] and the ubiquitous mitochondrial creatine kinase. Each of
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these enzymes play key roles in controlling energy availability.

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Cytosolic creatine kinase is a dimer that can be composed of muscle (M) or brain (B) type
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isoforms, creating three possible isozymes, the muscle specific MM-CK, the mixed MB-CK

found, for example, in heart and the brain specific BB-CK [29]. The cytosolic creatine kinase
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generates significant amounts of ATP upon brain activation and drives cognitive functions,
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dendrite formation and cell motility [47-50].

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Mitochondrial creatine kinase is the key enzyme to generate PCr from freshly synthesized

ATP. Octomeric mitochondrial creatine kinase assembles in a complex between inner and

outer mitochondrial membranes. This includes adenine nucleotide translocase and a porin,

which, when entered into the complex, favours transport of creatine over phosphocreatine.

The porin also allows efflux of high energy phosphate bonds from the mitochondrion in the

form of phosphocreatine, although ATP and ADP can also pass through porin in its anionic

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form. Creatine itself influences mitochondrial respiration rates, by altering the apparent KM of

the mitochondrial voltage-dependent anion channel for ADP [51]. Thus it is involved in

regulating oxidative phosphorylation [52, 53] most likely through controlling the availability

of ADP [54]. So called “creatine stimulated respiration” has been observed in muscle [55],

heart mitochondria [56] and in brain [51], likely through reduction in the dissociation

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constant for Mg-ATP with mitochondrial creatine kinase and creatine [55].

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In addition to its role in increasing mitochondrial ATP production rates, creatine is also

useful directly as an anti-oxidant, proving superior in some cases to glutathione [57, 58].

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Creatine has been suggested to be active at the GABA receptor [59, 60] although careful

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perusal of the original literature shows reported activity only for creatinine (the cyclic
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breakdown product) not creatine [61, 62]. The precursor, guanidinoacetate, is an agonist at

GABA-A receptors and an antagonist at GABA-ρ, in the latter case at least at physiologically
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relevant concentrations [7]. Creatine may also be active at the receptor responding to the
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major excitatory neurotransmitter glutamate, the NMDA receptor (NMDAR). Creatine has
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been implicated in population spike amplitude modulation [63] and in anti-immobility effects

in the tail suspension test [64]. The NMDAR polyamine binding site may be a possible site of
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activity [65]. This has led to the suggestion that creatine is a neuromodulator, with release of

creatine shown in response to electrical stimulation [59]; this release was blocked by absence
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of Ca2+ or by tetrodotoxin, and enhanced by blockade of K+ channels, consistent with

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neurotransmitter behaviour. Addition of creatine enhanced Na+K+ATPase activity via an

NMDA–calcineurin pathway [66]. Creatine is protective against glutamate toxicity, through

a mechanism unrelated to its ability to inhibit the activation of the mitochondrial permeability

transition pore [67]. In summary, the evidence that creatine modulates the NMDA receptor is

moderately strong but has not yet been proven conclusively.

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What is the evidence that creatine provides extra health benefits in the brain?

Creatine (8 g/day for 5 days, administered as 4 x 2 g each day) was given to 24 healthy young

volunteers (24 ± 9.1y) in a double blind placebo-controlled trial and their performance was

assesses on a serial calculation task, where subjects perform mathematical calculations for 15

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min, followed by a 5 min rest, then another 15 min of calculation. Performance on the latter

15 min gradually decreases in a manner that has been attributed to mental fatigue. The

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authors found that the performance decrement on the serial calculation task was decreased by

creatine supplementation , indicating that the capacity of the brain to perform a repeated task

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was enhanced by supplementation [68]. Similarly, 45 young adult vegetarians received 5

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g/day of creatine for six weeks in a placebo-controlled double-blind cross-over trial, with
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testing of their ability at backward digit span (a test of working memory) and Raven’s

Advanced Progressive Matrices [69]. Creatine was found to significantly improve their
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abilities at both of these tests; in the case of the Raven’s, equivalent to one standard deviation

or around 15 IQ points. Although tapping different cognitive domains, both of these tasks
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rely on speed of processing, potentially addressing the energy requirements in the “initial
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dip”. The finding of improved performance on backward digit span was subsequently
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confirmed in omnivores along with the observation of a decreased Blood Oxygen Level

Dependent (BOLD) effect on functional magnetic resonance imaging (fMRI) following a

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week of creatine supplementation (20 g/day for 5 days followed by 5 g/day for 2 days) [70].
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Conversely, a study giving ~2 g/day of creatine to 22 healthy young subjects (half received

placebo) for six weeks showed no significant change in any of the cognitive domains that

were measured, including memory and vigilance [71].

In general, the strength of evidence for creatine having positive effects on brain function in

healthy individuals is only moderate and it is plain that there is much further work to do. As

pointed out above, transport across the blood brain barrier is limited, reducing the

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effectiveness of oral supplements. The optimal dosing regimen of creatine remains to be

determined, practice effects should be controlled for [72] and the studies need to be

adequately powered. A cross-over design is helpful for controlling for baseline differences

between groups and studies would also benefit from measuring baseline brain creatine and

bioenergetics levels to determine whether baseline bioenergetics status is a significant

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variable in whether or not creatine supplementation has any efficacy in healthy subjects. It is

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known that bioenergetic status predicts cognitive performance [73]. Vegetarians have been

shown to have similar amounts of creatine in the brain as non-vegetarians [74], despite

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having lower muscle creatine [75].

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Studies where baseline bioenergetic status was possibly compromised include a 2006 study
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[76] which subjected 19 subjects to 24h sleep deprivation from 10 am, with tests of cognitive,

motor and mood function 6, 12 and 24h into sleep deprivation. Half of the subjects (N = 10)
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had taken creatine (20g/day in 4 x 5 g boluses for 7 days) and half (N = 9) placebo, with the

study designed to examine the effects of creatine on performance after sleep deprivation.
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Significant effects of creatine pre-supplementation were seen only at 24 h and were confined
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to choice reaction time, improved perception of fatigue and vigour, and working memory
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(Table 1). Interestingly, 24 h sleep deprivation has been shown to have no significant effect

on brain bioenergetics [77] although other authors have suggested that there may be
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individual differences in the response to sleep deprivation depending on baseline

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bioenergetics going into the period of sleep deprivation [78] and the creatine kinase system

has been shown to react differently according to the level of mental fatigue that is present

[79]. Certainly there is an energetic penalty to sleep deprivation that takes some days to

recover [77, 80].

The same group of researchers also examined the effect of creatine supplementation in

healthy elderly [81]. The creatine signal from the brain has been reported to increase with

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age, including increases in phosphocreatine and ATP [73, 82, 83] suggesting that total brain

bioenergetics capacity increases rather than decreases with age. With increased creatine

improving brain function, the reverse has also been shown, where memory exercises have

been shown to increase levels of creatine in the hippocampus of healthy elderly performing

training in the Method of Loci task, involving delayed recall, spatial and associative memory

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training, compared to elderly subjects who were merely undertaking tasks of everyday living

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[84].

The link between creatine and cognitive function in the aging brain has been further explored

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in old mice (C57Bl/6J, 24 months of age) which were supplemented with 1% creatine in their

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standard rodent diet from 12 months of age. Creatine fed mice showed longer “healthy” life
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span (9%), and longer total life span compared to controls. They also showed better

performance on object recognition and decreased latency in initiating exploration of a novel

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environment [85]. The animal model also allowed exploration of molecular effects, with

lower serum lactate in creatine fed animals. The authors also reported trends towards
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decreased accumulation of the aging protein lipofuscin, the accumulation of which has been
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shown in numerous studies to be due to the oxidative alteration of macromolecules by

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oxygen-derived free radicals [86], and decreased levels of 8-hydroxy-2ˈ-deoxyguanosine, a

marker of oxidative damage to DNA [85].

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Creatine supplementation in brain disorders

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Creatine is not currently used as a routine supplement in any human brain disorder apart from

deficiencies in creatine synthesis, although a number of clinical trials have been undertaken

in a range of different disorders, with varying results. Hampering the trials is lack of

information on the best dosage regime to increase brain creatine and an incomplete

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understanding of the longer term effects of creatine supplementation on endogenous creatine

synthesis and uptake.

Neurodegenerative disorders

Creatine, when trialled in human neurodegenerative disorders, has not lived up to the

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potential displayed in animal models (Table 2). A recent large Phase III trial of 10g/day in

Parkinson’s disease was discontinued due to futility [87]. Trials of creatine supplementation

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in Huntington’s disease have also proven disappointing [88, 89] although creatine use does

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reduce markers of oxidative damage [90] and lowers glutamate/glutamine levels [91].

Several possible explanations for the differences between mice and men have been essayed.

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These include the fact that the human trials did not use as much creatine as trials in mice and
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failure to fully understand the mechanisms of each disease [92]. The trials in Parkinson’s

disease which did show some promise were conducted in early stage Parkinson’s disease,
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while the Phase III trial was in patients whose disease was further progressed. Given the
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extent of damage to the brain that has already occurred in early diagnosed Parkinson’s
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disease [93], it might be more efficacious to trial use of creatine in as yet undiagnosed

patients who are at higher risk of Parkinson’s disease [94].

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Creatine supplementation has been suggested as possibly efficacious in early Alzheimer

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disease [95]; decline of brain creatine levels is associated with conversion from mild
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cognitive impairment to dementia [96] and there are known mitochondrial deficits and

decrease of BB-CK in frontal lobe in Alzheimer disease [97]. To date, no trial of creatine in

the dementias has been published.

Creatine in mental health

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Bioenergetic abnormalities are well documented in depression [98]. They are related to the

severity of the depression [99] and are normalised following treatment [100]. Direct current

stimulation, an emerging treatment for major depression, has been shown to modulate brain

bioenergetics, with the degree of response related to baseline bioenergetics status [44].

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Creatine supplementation has shown some promise in treatment of major depressive disorder

(Table 2). Several small open label trials have shown promise [99, 101] and a randomised 6

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week trial of creatine or placebo as an augmentation to escitalopram (serotonin transporter

inhibitor [102]) showed significant improvements in depression scores as early as two weeks

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after consumption [103]. Work in animals has shown a gender effect, with female rats

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responding to creatine in an anti-depressant type fashion [104]. Reflecting this, and the
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tendency of females to suffer more from depression [105], trials in humans have largely

focussed on females.
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A small open label trial of 4 weeks creatine supplementation in patients with chronic post-
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traumatic stress disorder showed significant improvements in ratings of disability, including

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improvement in sleep quality. The largest improvements were seen in those diagnosed with

comorbid depression [106].

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Creatine did not show efficacy in a small trial of persons with schizophrenia [107]. It is not
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possible to say from this work whether creatine may or may not have effects in this disorder.
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Bioenergetic abnormalities have been inconsistently reported in schizophrenia (reviewed in

[108]) and, as schizophrenia is a heterogeneous disorder, it is possible that a subset of

patients may benefit.

Creatine and neuroprotection

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There is considerable evidence in animals for neuroprotective effects of creatine against

traumatic injury [109], in ischaemia and in stroke. Creatine has been trialled as an open-label

administration of 0.4 g/kg/day in children with traumatic brain injury, with improvements

recorded in several clinical indices [110] and also in reported headaches, dizziness and

fatigue scales [111].

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Areas for translation to the clinic of creatine therapies

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Repetitive collapse of the upper airway during obstructive sleep apnea/hypopnea (OSA)

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exposes the brain of sufferers to frequent, transient, hypoxic episodes. Creatine levels, which

are significantly lower in the hippocampus of those with OSA [112] have been shown to be

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neurobiomarkers of disease severity and also of cognitive impairment [112]. Sufferers also
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show altered bioenergetics response to the hypopnea associated with apnea. Extended

hypoxia (12% O2, %satO2 = 86%) in healthy controls has been shown to have negligible
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impact on brain bioenergetics as measured with 31P magnetic resonance spectroscopy [113]
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but under similar levels of transient hypoxia during apnea, OSA sufferers display large
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increases in inorganic phosphate with decreases in ATP. Somewhat surprisingly, the creatine

kinase system, which in this case is most probably the cytosolic creatine kinase system,
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played no role, with no significant alteration in brain pH or in phosphocreatine [45].

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Creatine (and phosphocreatine) levels are known to be low in muscle of those with OSA and
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are increased by treatment with continuous positive airway pressure (CPAP) [114]. A trial of

creatine vs placebo on cognitive tasks undertaken in hypoxia (SpO2 reduced by 19%) showed

significant effects of creatine on tasks of complex attention in particular (Table 1). Scores of

executive function and cognitive flexibility were improved by creatine although the

difference did not reach statistical significance [115]. These are tasks which are typically

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impaired in OSA [116]. Taken together, this suggests that creatine may be of benefit in OSA,

particularly for the 50% of OSA sufferers who do not tolerate CPAP therapy.

Conclusion

In summary, the evidence for creatine as a nutraceutical for the brain is supportive of its use

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for cognitive enhancement, particularly in conditions where baseline bioenergetics are less

than optimal. Further trials are needed in neurodegenerative conditions, particularly at early

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stage when creatine may help to retard decline. Creatine may be of use in depression and

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depression-related disorders. Much work is still to be done in ischaemia, hypoxia and stroke

and the potential of creatine has yet to be tested in obstructive sleep apnea.

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We have yet to see the full potential of von Liebig’s discovery.
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Acknowledgements
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This work was supported by a grant from the Australian NHMRC ( Fellowship to CR
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#630516).
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Table 1. Outcomes of studies testing effects of creatine in healthy humans

Supplement Subjects Tests Outcome Ref.

8 g/day for 5 24 healthy Serial calculation task Significant [68]
days, volunteers (24 ± improvement
administered 9.1y)
as 4 x 2 g each
day. Double

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blind placebo
controlled
5g/day for 6 45 healthy Backward digit span Significant [69]
weeks vegetarians, 12, Raven’s Advanced improvement

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Double-blind, males, 33 females Progressive Matrices Significant
placebo improvement

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controlled
cross-over
design
20 g/day for 5 22 healthy Backward digit span Significant [70]

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days followed volunteers improvement
by 5 g/day for Age not specified Raven’s Advanced Non-significant
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2 days Progressive Matrices improvement
BOLD response to Significant decrease
flashing chequerboard in BOLD response
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0.03 g/kg/day 22 subjects (21 ± Simple reaction time No significant [71]

for 6 weeks 2 y) Code substitution change in any of the
Double-blind, (+delay) tasks measured
placebo Logical reasoning
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controlled symbolic
Mathematical
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processing
Running memory
Sternberg memory
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recall
20g/day for 5 121 female Word recall Vegetarians showed [117]
days as 4 x 5g subjects 20.3 (SE sig improvement
throughout day 2.1) y Omnivores got
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Placebo 51 omnivores worse

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controlled 70 Reaction time No effect on reaction

vegan/vegetarians time. Performance
less variable on
creatine
Vigilance No effect
Verbal fluency No effect
5g/day for 15 34 subjects 21 ± Memory scanning Some significant [118]
days of 1.38 y Number pair matching improvements
creatine No vegetarians Sustained attention recorded, depending
ethylester Arrow flankers on the statistical test
Double-blind IQ (modified Raven’s) used
placebo

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controlled
5 g x 4/day for 19 subjects 21.1 Sleep deprivation with [76]
7 days ± 1.85 y serial testing at 6, 12
and 24h of:
Working memory Significant
(Random movement improvement at 24h
generation)
Forward/backward digit No significant
span differences

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Spatial memory (Corsi No significant
Block) differences
Psychomotor (choice Significantly better

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reaction time) at 24 h
Mood state (POMS) Decreased fatigue
and increased vigour

SC
at 24h
7 days, 15 subjects 31y Trial of creatine in [115]
20g/day (4 x (21-55 y) 10 oxygen deprivation
5g) males 5 females (10% O2)

U
Randomised, Complex attention Creatine improved
double-blind, scores under hypoxia
AN
placebo compared to placebo
controlled Motor cortical Increased under
cross-over excitability hypoxia compared to
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design placebo

5g creatine Healthy elderly Tested at baseline, 7 [81]

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monohydrate x 76.4 (8.48 y) N = days and 14 days,

4/day or 15 & 17. Measured change in
TE

placebo. Group performance ∆: Not significant

1 7 day Random number
placebo then 7 generation Forward recall
day creatine, Forward/backward digit improved
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Group 2 14 recall Forward and

day placebo. Spatial memory (Corsi backward recall
Block) improved
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Significant
Delayed memory task improvement
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4 x 5g for 5 Healthy elderly Mini Mental State Not significant [119]

days then women N = 56 Exam
5g/day for total assigned to 4 Stroop Not significant
of 24 weeks. groups (Creatine Trail making Not significant
Randomised Cr or placebo PL Digit span Not significant
placebo- with or without Delay recall Not significant
controlled, strength training Geriatric depression Not significant for
double blind ST. index creatine but
trial of creatine Cr N = 13 improvement with
with or without PL N = 12 strength training.
strength Cr + ST N = 12
training. PL + ST N = 10

18
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Table 2. Outcomes of studies testing effects of creatine in brain disorders

Supplement Subjects Tests Outcome Ref

2 yr study Parkinson’s SPECT No significant change [120]
20g/day for 6 disease N = 60 Dopamine binding No significant change
days, then 40 received UPDRS No significant change
2g/day for 6 creatine, 20 overall - depression score
months, then placebo. Study significantly improved in
4g/day for total finished with creatine group

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time of 2 years. N = 31 and 17, Increase over time Threefold smaller
Placebo respectively in dopaminergic increase in dopaminergic
controlled, therapy therapy over 2 years in

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randomised creatine group
10g/day for Parkinson’s Global statistical No change in primary or [87]
minimum of 5 disease N = test using 5 secondary outcome

SC
years. Double- 1741 within 5 measures of measures. Trial
blind, parallel years of Parkinson’s disease terminated due to futility
group placebo Parkinson’s progression
controlled trial. disease

U
diagnosis.
Placebo, 61.5
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(9.6) yrs
Creatine, 62.1
(9.7) y.
M

1 yr study Huntington’s UPDRS No significant effect of [89]

5g/day (1g, 3g disease. N = Battery of creatine was found
then 1 g with 26 creatine cognitive tests
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each of the three (50.1 ± 2.6y)

meals) and N = 15
TE

Placebo placebo (49.6

controlled. ±1.9 y)
Assigned by
independent
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investigator
16 week Huntington’s UPDRS No change [90]
tolerability trial disease. N = Brain creatine ↑ 13% occipital lobe
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8g/day (2 x 4 g 64 levels ↑7.5 % frontal lobe

each day) for 16 Creatine age Serum 8OH2`dG Significantly less than
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weeks. 44.7, placebo levels placebo

Randomised, 47.9 y.
placebo-
controlled.
10 month trial Huntington’s Total motor score No change [88]
10g/day for 10 disease. N = UPDRS
months 13 creatine, N Brain creatine No change
= 4 spousal levels Significantly elevated
controls
1
8-10 weeks Huntington’s H MRS (Glx Significantly lower [91]
creatine (20 disease. resonance) compared to baseline
g/day for 5 days N = 16 UPDRS No change

19
 ACCEPTED MANUSCRIPT
then 6/day with MMSE No change
none on
Sundays (to
prevent
augmentation)
10 week trial of Major Hamilton Significant improvement [103]
creatine vs depressive Depression score over placebo as early as
placebo as disorder Montgomery- week 2
augment for Escitalopram Åsberg Depression Significant improvement

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escitalopram + either: Rating Scale over placebo as early as
3g/day for one N = 25 week 2
week then creatine

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5g/day for 7 45.7 (127) y
weeks N = 27
placebo

SC
47.5 (9.5) y
All female
3 month Schizophrenia Positive and No significant effect [107]
randomized N = 10, 7 negative syndrome

U
cross over trial males, 5 scale (PANSS)
3-5 g/day females Clinical Global
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creatine or 42.8 ± 8 y. Impressions
placebo Cognitive test No significant effect
battery
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No significant effect
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Open label trial Post Clinician Improvement in all [106]

3g/day for one traumatic administered PTSD scores, intrusiveness
TE

week then stress disorder scale most improved,

5g/day for 3 N = 10 avoidance the least.
weeks. Hamilton Rating
Scales for
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Depression Significant improvement

& Anxiety No improvement
Clinical Global Significant improvement
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Impressions
Sleep Quality Scale Improved
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Sheehan Disability No significant change

Scale
Open label trial Traumatic Clinical outcomes Improvements seen in [110]
of creatine, brain injury several variables
randomized to N = 39,
study and children aged Headaches Significantly less [111]
control groups 1-18 y Dizziness Significantly less
0.4 g/kg/day for Fatigue Signficantly less
6 months or
nothing

20
 ACCEPTED MANUSCRIPT

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123. Kornak, J., D.A. Hall, and M.P. Haggard, (2011) Spatially Extended fMRI Signal

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Figure Captions

Figure 1. Synthesis and uptake of guanidinoacetate and creatine and their deficiency

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syndromes. AGAT, L-arginine-glycine amidino transferase; GAMT, guanidinoacetate-
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methyltransferase; SLC6A8, solute carrier 6A8.
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Figure 2. Current understanding of metabolism, compartmentation and synthesis of

creatine and its analogues in the brain. Creatine synthesis and uptake is different in
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subpopulations of cells in the brain [121]. Some neuronal, astrocytic and oligodendrocyte
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subpopulations contain both enzymes of the creatine synthesis pathway (designated 1 in the
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figure) AGAT (Arginine-glycine aminidino transferase) and GAMT (Guanidinoacetate

methyl transferase). Others, (2) possess only AGAT, and are therefore net producers of
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guanidinoacetate (GAA), or possess only GAMT and therefore need the creatine transporter
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SLC6A8 in order to take up the precursor GAA. A third subpopulation (3), possess none of

the synthetic enzymes and are absolutely reliant on SLC6A8 to take up creatine produced by

other cells (Type 1 or 2). A fourth subpopulation that does not possess any synthetic enzymes

or transporters and does not use creatine (4). SaM; S-adenosylmethionine; SaHC, S-

adenosylhomocysteine. Figure adapted from [122].

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Figure 3. Timecourses of substrate availability following stimulation in brain. Panel A

shows timecourse of phosphocreatine changes in occipital lobe following a 4s flashing

chequerboard stimulus. Data were collected from a single human subject at 1.5 Tesla using an

8 cm 31P surface coil with one single transient collected every 2s. The duty cycle (4s 16 Hz

photic stimulation followed by 26 s of rest) was repeated 8 times and spectra were binned to

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make a timecourse with 2 s resolution with each spectrum comprising 8 scans [42]. Dotted

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lines show 95% confidence interval of the fitted function. Panel B shows the generic blood

oxygen response to a stimulus detected using functional magnetic resonance imaging

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(adapted from [123]). Panel C shows the glucose concentration at a fixed point in the cerebral

cortex of rat during and following a single wave of spreading depression (induced by

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application of KCl) (adapted from [36] with the permission of the authors).
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Figure 4. Creatine, cytosolic and mitochondrial compartments, their respective creatine
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kinase isoforms and interacting enzymes. Cytosolic creatine kinase (CK) interacts with

ATP produced via glycolysis. It acts as a rapid supplier of ATP for hydrolytic cytosolic
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reactions, including the Na+K+-ATPase. Mitochondrial creatine kinase (MiCK) forms a

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metabolon with a porin (a voltage-dependent anion channel), which when combined with
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mitochondrial creatine kinase prefers a cationic form favoring creatine influx. These proteins

also combine with the adenine nucleotide translocase, ATP-synthaseF0F1 and an inorganic
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phosphate carrier. This metabolon is visible on electron micrographs [124]. Deletion of one
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or the other forms of CK has metabolic and behavioural consequences

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